While the therapeutic effect of opioids analgesics is mainly attributed to µ-opioid receptor (MOR) activation leading to G protein signaling, their side effects have mostly been linked to β-arrestin signaling. To shed light on the dynamic and kinetic elements underlying MOR functional selectivity, we carried out close to half millisecond high-throughput molecular dynamics simulations of MOR bound to a classical opioid drug (morphine) or a potent G protein-biased agonist (TRV-130). Statistical analyses of Markov state models built using this large simulation dataset combined with information theory enabled, for the first time: a) Identification of four distinct metastable regions along the activation pathway, b) Kinetic evidence of a different dynamic behavior of the receptor bound to a classical or G protein-biased opioid agonist, c) Identification of kinetically distinct conformational states to be used for the rational design of functionally selective ligands that may eventually be developed into improved drugs; d) Characterization of multiple activation/deactivation pathways of MOR, and e) Suggestion from calculated transition timescales that MOR conformational changes are not the rate-limiting step in receptor activation.

This work was supported by National Institutes of Health grants DA026434, DA034049, and MH107053(to M.F.) and by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 675451 (CompBio Med project; to G.D.F). Computations were run on resources available through the Scientific Computing Facility at Mount Sinai, the Extreme Science and Engineering Discovery Environment under MCB080077, which is supported by National Science Foundation grant number ACI-1053575, and the high-throughput molecular dynamics (HTMD) platform.